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DNA Nanotechnology for Precise Control over Drug
Delivery and Gene Therapy
Chava Angell, Sibai Xie, Liangfang Zhang, and Yi Chen*
From the Contents
1. Introduction ........................................ 1118
2. DNA Nanotechnology ........................... 1118
3. DNA-Based Approaches for
Drug/Gene Delivery ............................. 1120
4. Summary and Future Outlook............... 1129
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DNA Nanotechnology
Nanomedicine has been growing exponentially due to its
enhanced drug targeting and reduced drug toxicity. It uses
the interactions where nanotechnological components
and biological systems communicate with each other
to facilitate the delivery performance. At this scale, the
physiochemical properties of delivery systems strongly
affect their capacities. Among current delivery systems,
DNA nanotechnology shows many advantages because
of its unprecedented engineering abilities. Through
molecular recognition, DNA nanotechnology can
be used to construct a variety of nanostructures with
precisely controllable size, shape, and surface chemistry,
which can be appreciated in the delivery process. In this
review, different approaches that are currently used for
the construction of DNA nanostructures are reported.
Further, the utilization of these DNA nanostructures with
the well-defined parameters for the precise control in drug
delivery and gene therapy is discussed.
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1. Introduction
Naked therapeutic agents, which include small-molecule and
biomolecular drugs, have some intrinsic issues that prevent
the full realization of their functions in the body. This includes
poor solubility, poor stability against chemical and enzymatic
degradation, inability to pass the biologic barriers, unwanted
side effects and toxicity. To overcome these challenges, many
different drug delivery systems have been developed in the
past decades.[1,2] They can be generally classified into two
categories: natural and biomimetic delivery systems such
as virus[3] and red blood cells mimicking carriers,[4] and
synthetic delivery systems such as liposomes and polymeric
particles.[5,6] These delivery platforms use natural or synthetic
particles to carry drugs and thus bypass their physiochem-
ical limitation and barriers for effective delivery. Through
appropriate parameter design, these delivery systems can
effectively increase the drug loading efficacy, the circulation
time in body, and improve the final therapeutic results.[7,8]
Many of them have entered clinical trials and some have
been approved for clinical use.
Although these drug carriers are actively being
developed, there is still a measurable gap that has to be
surmounted to achieve the ultimate goal of drug delivery:
the maximal therapeutic efficacy with minimal toxic effects.
In spite of different routes of administration, including
oral, injection, transdermal and inhalation, the delivery
systems and the therapeutic cargos can be traced to the cel-
lular level. This stimulates the exponential development of
nanomedicine which uses nanotechnology tools to enhance
drug targeting, reduce drug toxicity, and enable reliable
diagnosis.[9,10] Among these newly developed delivery sys-
tems, DNA nanostructures show high promise. DNA is
a biopolymer that can self-assemble into double helices
through by Watson–Crick base pairing and is stabilized by
hydrogen bonds, p–p stacking, and hydrophobic interac-
tions. They have well-defined structures: the helical turn is
3.4 nm and the diameter is ≈2.2 nm for the B-form double-
helical molecules. Fueled by this base-pairing ability, DNA
nanotechnology has enabled many strategies to form DNA
nanostructures with well-controlled size, shape, and surface
chemistry. In nanomedicine, these properties strongly affect
the delivery capacities.[11,12] and thus give DNA nanostruc-
tures unique control abilities to address some of the unmet
delivery requirements.
This review will discuss the applications and weigh the
usefulness and viability of DNA nanostructures in precision
nanomedicine. The discussion will begin with an introduc-
tion to DNA nanotechnology, including some of the types of
structures that can be formed. Following this will be an elu-
cidation of the DNA oligonucleotide approach, the original
strategy used to create DNA nanostructures, and current uses
of this strategy for drug delivery. The next motif that will be
discussed is the DNA origami strategy and what has currently
been achieved with DNA origami in drug and gene therapy
as well. Finally, we will touch on some of the unique shapes
and modalities that have been created for the purpose of
DNA drug delivery and finish with our remarks and thoughts
on the future viability of this field.
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2. DNA Nanotechnology
DNA Nanotechnology is a branch of nanoengineering that
utilizes the precise and predictable nature of complemen-
tary DNA base pairing to create 2D and 3D structures. This
branch of nanotechnology has many applications in medicine
and biotechnology as DNA can easily be functionalized with
binding ligands and fluorescent dyes for signal readout.[13]
The field was pioneered in the 80’s by Ned Seeman, who pro-
posed the construction of an immobile 4-way arm junction
comprised of DNA and the connecting networks through
sticky ends.[14]
After a few decades of development, this field has been
exponentially explored. Varies of DNA nanostructures and
applications have been successfully reported. Recently, DNA
nanostructures start to be used as a creative solution to both
the accumulation and off target effects of other drug delivery
modalities as it is a nontoxic, biologically derived material
that can be easily functionalized to target receptors on the
cell surface and can also be covalently or electrostatically
attached to metallic nanostructures.[15]
To create stable structures out of DNA, certain sequence
symmetry has to be minimized to prevent the instability that
is seen in many intermediate structures, such as Holliday
junctions (Figure 1A).[14] There are also many design consid-
erations such as the incorporation of sticky ends, and recip-
rocal exchanges for stability, also known as ‘crossovers’.[16]
DNA nanotechnology is a highly intriguing strategy for
self-assembled drug delivery structures as it allows for the
creation of precise structures that can mimic nanoparticles or
viral capsids. Such structures include polyhedra of different
sizes and shapes such as cubes,[17] tetrahedra,[18–20] octa-
hedra,[21] buckyballs,[19] and icosahedra.[22,23] Larger shapes
based on the DNA origami method are also reported.[24]
Goodman et al. reported a facile way of building DNA tet-
rahedra using four different strands, with the struts less than
10 nm in length.[18] Under the simple process of cooling from
95 °C to 4 °C in 30 s, tetrahedra form under a yield of 95% and
can be characterized by a single band on a native gel.
Another approach was proposed by He et al. to construct
tetrahedra, dodecahedra, and bucky ball structures through
DNA tiles.[19] The proposed three-point star motif was used
for 2D crystal formation.[25] In this approach, the flexibility
of the long central strand was enhanced by elongating the
non-binding hinge parts which they call “loops”, so that the
motifs tend to bend and form closed structures. The concen-
tration of the motif also affects the resulting structure. A loop
length of 5 bases and a low concentration of 75 nm gives a
final result of tetrahedra, while 3 bases loops and 50 nm gives
dodecahedra, and 3 bases loops and 500 nm concentration
gives buckyball structure. Each strut is around 14 nm.
C. Angell, S. Xie, Prof. L. Zhang, Prof. Y. Chen
Department of NanoEngineering
University of California
San Diego, La Jolla, CA 92093, USA
E-mail: yic047@ucsd.edu
DOI: 10.1002/smll.201502167
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Figure 1. Demonstration of DNA nanostructures. A) immobile 4-strand junction proposed by Seeman. Reproduced with permission.[14] Copyright
1983, Cell Press. B) Icosahedral structure formed by flexible five-point-star motifs. Reproduced with permission.[22] Copyright 2008, National
Academy of Sciences. C) Truncated cubic structure. Reproduced with permission.[17] Copyright 1991, Nature Publishing Group. D) Different
structure formed by the same three-point-star motif with different “loop” length and concentration. Reproduced with permission.[23] Copyright
2009, Wiley-VCH.[23] E) Single-stranded DNA bricks form 3D canvas. Reproduced with permission.[27] Copyright 2012, American Association for the
Advancement of Science (AAAS).

Similar tile approach can also be used to form icosahe-
dral DNA nanostructure (Figure 1B).[22] The same group
reported in 2008 a method to fabricate truncated icosahedra
of diameter ≈20 nm. The five point star motif used in this
method has a four base loop. They also reported that the
concentration of the motif also has an effect on the predomi-
nant structure that can form: the samples form a big cage
structure (≈115 nm in diameter) under higher concentration.
The icosahedra sample was prepared in the concentration of
20 nm to prevent the formation of larger structure. DNA
nanostructures can also be formed through step-by-step pro-
cess. Chen et al. demonstrated this method by constructing a
nano-scale cube structure (Figure 1C).[17] In each step they
hybridize two squares and in the last step the ends are con-
nected to form a cube. Bahtia et al. reported a three-step
way to encapsulate gold nanoparticles into a truncated DNA

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nanostructure (Figure 1D).[23] They first anneal DNA strands
to form five-arm vertex motifs, and then combine the two
“halves” form icosahedron with complementary binding sites.
The two halves were put into a gold nanoparticle solution.
The icosahedron with diameter of roughly 19 nm can success-
fully encapsulate 2–3 nm diameter gold nanoparticles. Other
icosahedra have been reported that can intercalate doxoru-
bicin (Dox).[26]
An important concept in DNA nanotechnology is ease
of modular self-assembly. In 2012, Ke et al. reported the con-
struction of short “DNA bricks” that can be used to create
many different types of structures without having to redesign
staple strands and scaffolding routing as is necessary in cre-
ating varying origami structures (Figure 1E).[27] The authors
reported essentially a lego-like modality in which each brick
had a distinct shape and could bind to 4 neighbors just as a
lego can fit into specific holes. These interactions are defined
by the domains of the DNA bricks which have unique
sequences. Therefore, each 32 nucleotide brick is distinctive
and can be combined to create different 3D structures with
both controllable surface features and precise cavities within
the structure. 102 different structures were created using this
method merely by selecting the bricks needed which greatly
simplifies the process of creating 3D DNA nanostructures.[27]
There are several methods to make viable, stable struc-
tures comprised mostly or entirely of DNA: DNA origami,
oligonucleotide design, and rolling circle amplification. In
this review, we will touch on the methodologies used to
form DNA nanostructures and how those methodologies are
being coupled with both gene silencing therapies and cancer
therapeutic drugs to more successfully deliver and control
the delivery of therapeutic modalities to site specific cells
in vivo.
3. DNA-Based Approaches for Drug/Gene
Delivery
There are many methods that have been coupled to DNA
nanotechnology to effectively deliver therapeutic modalities,
including drug modalities, gene-silencing modalities, and the
incorporation of cellular targeting into DNA nanostructures.
Many DNA drug delivery systems include the incorporation
of doxorubicin (Dox), an anticancer therapeutic,[28,29] as the
proof of concept for small molecular drug delivery. Dox can
intercalate into the DNA structure and allow it to form prop-
erly by relieving torsional stress, as showed in Figure 2.[29–31]
However, despite Dox’s potency as an anticancer drug, it
can have serious side effects when it accumulates off-target
such as cardiomyopathy, which can lead to congestive heart
failure.[32] Therefore, avoiding off-target or premature
delivery must be achieved.
Another therapeutic technique that many researchers
have been taking advantage of is gene-silencing through RNA
interference.[33] Small interfering RNA (siRNA) in particular
has been utilized by many researchers for therapeutic gene
silencing and can be modified to be target specific to avoid
off target silencing effects.[34] RNA interference can usually
be used without triggering the immune system. However,
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Figure 2. Intercalation of Doxorubicin. Reproduced with permission.[31]
Copyright 2014, Elsevier.
the introduction of the artificial siRNA can monopolize the
natural interference pathway of the RNA silencing complex,
which is an important part of the cellular function.[35] One
of the biggest challenges in RNA delivery is stability in vivo,
but chemical modifications of the oligomers can increase the
stability.[36]
The targeting issue using DNA nanostructures can be
achieved through the use of DNA aptamer sequences and
also through the use of functionalizing the DNA with tar-
geting ligands and can help avoid many of the undesirable off
target effects.[32,37,38] For example, Zhu et al. created synthetic
DNA adducts to allow for targeted anticancer delivery.[32]
These take advantage of the overexpression of certain recep-
tors during cancer.[39]
Once been uptake by cells, DNA nanostructure can be
designed to respond to environmental triggers to release
loaded drugs, such as incorporation of linkers that are
responsible to changes in the pH.[29,30,40,41] This technique can
also aid in mapping the pH changes inside the cells itself, as
reported by Modi et al.[41]
There are several possible pathways for these DNA
nanostructures to enter the cell and partially be explored by
using inhibitors to block certain pathways.[42,43] These path-
ways can be divided into two types: endocytotic pathways
and non-endocytotic pathways.[44] The endocytotic pathways
include phagocytosis, clathrin mediated endocytosis, caveolae
mediated endocytosis, and macropinocytosis. However, it is
not well understood what mechanism is responsible for the
successful uptake of DNA nanostructures. Preliminary data
such as that demonstrated by Chen et al. suggested clathrin
receptor mediated endocytosis.[43] They measured the mecha-
nism of the uptake of their rolling circle amplification (RCA)
nanoribbons through inhibitors such as chlorpromazine, a
clathrin inhibitor, genistein, a caveolae mediated inhibitor,
methyl-β-cyclodextrin, a lipid-raft endcytosis inhibitor, wort-
mannin, a macropinocytosis inhibitor, and NaN3, an energy
dependent endocytosis inhibitor. Final results suggested
that the uptake is clathrin mediated and lipid raft mediated
endocytosis.
Chen et al. also studied the mechanism of transfection of
DNA nanoparticle (DNPs) and suggested that nucleolin is a
good target for non-viral gene delivery with the presence of
glucocorticoid receptor (GCR).[45] Besides the disruption of
a raft protein, flotillin also decrease the transfection of DNPs,
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while inhibition of other endocytic pathways barely affects
the process.
Most of the cell membranes carry net negative charge
and nucleic acids also carry a net negative charge.[46] As a
result, some DNA nanostructures, not containing targeting
modalities, may need some kind of cationic transfection
agent, such as Lipofectamine, to increase the cellular uptake
and therapeutic efficacy.[46] Positively charged intercalators
or positively charged proteins, such as Dox, viral capsule
proteins, or cationic polymers, may also help reduce the net
negative charge thus reducing the electrostatic repulsion in
the DNA structure itself and between the DNA nanotech-
nology and the cell membrane.[33,47–49]
For the purpose of this review, we will discuss the
different mechanisms of DNA nanostructure formation as
separate categories. These mechanisms are integral to what
can be achieved for the shape and size of the structures and
can affect what can be delivered and the release kinetics of
delivery. Some large structures can only be created through
the use of DNA origami and certain modularity can only be
achieved using the oligonucleotide approach. However, some
structures are hybrids of different fabrication methods and
may fit multiple synthesis categories. Therefore, they will be
categorized by their primary method of synthesis.
3.1. Oligonucleotide Approach
Due to the precise and predictable pairing nature of DNA
chains, the size and shape of the nanoscale DNA constructs
can be designed to fit current drug delivery needs. Moreover,
overhangs can be introduced on to the structures to allow
easy functionalization of fluorescent dyes, targeting agents or
even drugs.
The oligonucleotide strategy was reported first by
Nadrian Seeman in 1983 and is widely considered the foun-
dation of DNA nanotechnology. He synthesized a stable
4 arm junction with 4 sixteen base strands by minimizing the
strand symmetry.[14] The oligonucleotide strategy requires
more design consideration as there are no viable programs
for designing these structures. Oligonucleotides have been
used to create many types of structures such as nanotubes,[50]
tetrahedra,[13,51] icosahedra,[23] and many other polyhedral
structures.[19,21]
In 2010 Kim et al. reported a simple method of inter-
calating Dox into DNA tetrahedra as carriers to reverse
drug resilience of cancer cells.[13] The dye Cy5 was incor-
porated into the tetrahedron for characterization purpose
(Figure 3B). Results show that even without any targeting
agent, the DNA tetrahedra are able to enhance uptake and
bypass the efflux process in multidrug resistant breast cancer
cell line (MCF-7 cancer cells). These results suggest certain
shape dependence on transfection efficacy.
An example of DNA polyhedra assembled from oligo-
nucleotides was reported by Chang et al. in 2011.[26] They
designed a six-point-star motif with five sticky ends that are
self-complementary and one end with targeting aptamer to
assemble into icosahedra with diameter of around 28 nm,
while the ends with aptamer erect. Dox was intercalated
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into the structure without affecting the shape. The Mucin-1
(MUC-1) aptamer was used to target MUC 1+ MCF-7 cells.
The dox–apt–icosahedron complex shows great specificity
and aptamer-mediated internalization for killing epithelial
cancer cells.
DNA polyhedral nanostructures have also been used in
gene delivery. Lee et al. reported a way to deliver siRNA
using a tetrahedral DNA structure with a hydrodynamic size
of 28.6 nm with narrow distribution (Figure 3C).[52] Molec-
ular identical oligonucleotide particles (ONPs) with six folic
acid ligands per particle were injected in nude mice with
human epidermoid carcinoma cell (KB) xenograft tumors.
Results show that the ODNs can accumulate primarily in
the tumor and kidney. The circulation time of the ODNs
is also a lot longer than the parent siRNA (blood half-life
t1/2 ≈ 24.2 min vs 6 min).
A concept of nanotrain was reported by Zhu et al. in
2013.[53] Two strands of ODNs alternatively bind to each
other triggered by an aptamer trigger to form a longer chain
train-like structure with an aptamer sgc8 on one end to target
specific cells (Figure 3D). Dox is intercalated into the DNA
double strand. The complex can be specifically transport the
drug to target cancer cells, initiate endocytosis and unload
the drugs within the cells. The complex was tested in vivo
in NOD.Cg-Prkdc (scid) IL2 mice and showed reduced side
effects and stronger therapeutic potency.
Wu et al. reported a modular aptamer based DNA
nanoassembly (AptNA) approach (Figure 3E).[54]
Multifunctional DNA sequences are first assembled into
Y-shaped functional domains and then connected to
X shaped connectors. The connector has a photopolymeriz-
able arm so that the building unit with different functional
domains could be polymerized into spherical structure of
diameter around 200 nm. The aptamer sgc8 was used to
target CCRF-CEM (T cell acute lymphoblastic leukemia
cell line) cancer cells. The cells showed increased viability
compared to the treatment by free Dox. The DNA nanopar-
ticles also showed selectivity on CEM cells rather than the
non-target Ramos cells.
Li et al. developed a tetrahedron motif bearing unmethyl-
ated cytosine–phosphate–guanine (CpG) motifs which have
immunostimulatory properties.[55] This is due to the fact that
these motifs can bind to certain cellular receptors known as
endosomal Toll-like receptor 9 and trigger an immunostim-
ulatory response. After incubation with macrophage cell
line (RAW264.7 cells), these structures were mostly local-
ized in the cytoplasm as imaged by confocal microscopy.
Degradation studies were also conducted by incubating the
tetrahedrons in fetal bovine serum (FBS). These structures
remained stable at 4 hours whilst simple DNA duplexes
degraded in the span of 2 hours.
Sellner et al. reported in 2015 an in vivo study of gene
delivery by DNA nanotubes. The nanotubes were assem-
bled by the single stranded tile (SST) method, in which each
tile consists of four 10–11 bases domains and a total length
of 42 bases.[50] Sticky ends of poly-As stick out of the tube
side walls for functionalization. CpG and fluorescent dyes
ATTO488-dUTP or Cy3-dUTP were conjugated onto the
structure. The in vivo study on anesthetized mice showed
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Figure 3. A) The original DNA tetrahedral structure proposed by Turberfield. Reproduced with permission.[18] Copyright 2005, AAAS. B) Modified
DNA tetrahedral structure with Cy5 dye and Dox intercalation. Reproduced with permission.[13] Copyright 2013, Royal Society of Chemistry.
C) Incoporation of siRNA onto the struts of the DNA nanotetrahedron. Reproduced with permission.[52] Copyright 2012, Nature Publishing Group.
D) Proposed drug delivery route of DNA nanotrain.[53] E) Multifunctional aptamer-based nanoparticles. Reproduced with permission.[54] Copyright
2013, American Chemical Society.

that while plain nanotubes do not induce an immune
response, CpG conjugated nanotubes can target tissue-res-
ident macrophages and reach intact muscle tissue almost
immediately, and elevate immune response strongly. The
DNA nanotubes also had some stability against DNase I
within muscle tissue.
3.2. Origami Approach
As seen above, DNA oligonucleotides allow for the crea-
tion of precise structures that can be manipulated to deliver
both gene therapies and anticancer therapeutics. Other
than that, other strategies for synthesizing precision DNA
nanotechnology are also extremely attractive for their shape
control, control of flexibility and size, and ability to create
larger, modular structures that can even be extended to
include kinematic, movable joints.[27,56,57]
This approach is normally called DNA origami. It was
pioneered by Paul Rothemund in 2006 (Figure 4B).[58] DNA
origami refers to the folding of a long stranded bacteriophage
(7240 nt) through the use of over 200 complementary staple
strands to fold the backbone. Paul Rothemund used the
M13m18 bacteriophage and his original structures were 2D.
Each oligonucleotide is considered a 6 nm pixel to build any
pattern of desire. By putting sticky ends on the connection

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Figure 4. A) DNA octahedron obtained by folding a 1.7 kilobase backbone. Reproduced with permission.[63] Copyright 2004, Nature Publishing
Group. B) Rothemund’s DNA origami shapes. Reproduced with permission.[58] Copyright 2006, Nature Publishing Group. C) 3D DNA origami
structures. Reproduced with permission.[60] Copyright 2009, Nature Publishing Group. D) Large 3D structures synthesized by Ilumina to carry
therapeutic loads. Reproduced with permission.[24] Copyright 2014, AAAS.

part of each construct, an extended array can also be formed.
Since then, the DNA origami motif has been extended to
three dimensions.[59,60] There are now several programs
that facilitate the design of DNA origami structures such as
caDNAno and SARSE.[56,61] With DNA origami, structures
such as smiley faces, tetrahedrons, DNA nanotubes, DNA
barrels, DNA “dolphins” and many other shapes are pos-
sible.[20,28,56,58,62] Many groups have worked on extending this
modality and facilitating its use for structural formation and
drug delivery.
Shih et al. reported back in 2004 an idea similar to the
later developed DNA approach, which is to fold a long
backbone DNA chain by binding it at different points using
staple strands (Figure 4A).[63] In this example a 1669-nucle-
otide single-stranded DNA is used as the backbone and
five 40-nucleotide strands are used to fold the backbone.

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Double-crossover and paranemic-crossover structures are
designed to ensure the stiffness of the structure. It was
proposed that the long backbone chain can be readily
amplified.
Douglas et al. reported that using the same DNA ori-
gami method, it is also possible to fabricate 3D structures.[61]
They used honeycomb-pleat-based strategy to bring the
helices together at a certain angle to construct 3D struc-
tures (Figure 4C). They also developed a software called
caDNAno, an easy tool to design 3D structures and program
the staple strands. The whole design and synthesis process
took 2 weeks as they reported.
One of the largest challenges that faced DNA origami
was the time it takes to fold and hybridize the structures. In
2012, Sobczak et al. determined that a long annealing pro-
cess is not necessary to create these structures in a “one-pot”
reaction scheme as there is a distinct range where DNA will
fold and hybridize.[64] These experiments also reported that
DNA folding and unfolding are non-equilibrium processes as
the temperature for unfolding is higher than the one at which
the structures formed. This experiment allows DNA origami
to be on a more feasible time scale.
There is the desire within this field to create large struc-
tures that mimic viral capsids. Iinuma et al. reported the con-
struction of polyhedra from million daltons (MD) monomers
(Figure 4D).[24] These monomers created a 5-MD DNA ori-
gami three arm junction that was sixty times more massive
than previously reported 3-arm motifs. These junctions have
a “dynamic connector” design and Iinuma et al. created a
DNA tetrahedron, a cube, a triangular prism, a pentagonal
prism, and a hexagonal prism. All structures were on the
order of 20–60 MD and had compartments that are similar
in size to those of bacterial microcompartments and could be
used to carry therapeutic loads.
In drug delivery, the kinetics and controllability of
release is an important factor for consideration. If the thera-
peutic is released in a burst mechanism as opposed to a slow,
tunable release, there will be a higher initial delivery, how-
ever this can lead to high physiological damage and a lower
lifetime of the therapy, which leads to the need for higher
dosages.[65] DNA origami allows for either controlled or trig-
gered release of a therapeutic modality through either the
intercalation of positively charged molecules or the linking
of certain peptides or proteins onto the surface of the DNA
origami structure.
In 2012, Zhao et al. designed and fabricated a structure
that has controllable release properties of Dox, a well-known
anticancer therapeutic. These structures take advantage of
the fact that DNA origami structures with a specific twist
density will only correctly fold in the presence of an interca-
lating molecule such as Dox (Figure 5A).[28] This was origi-
nally demonstrated by Ke et al. who inserted base pairs (bp)
in between crossover junctions.[47] These insertions, while
relieving electrostatic repulsion, also increase torsional strain,
thus destabilizing the structure and allowing for the inter-
calations to bind to the structure.[47] This binding relieves
the stress and encourages the correct folding of the origami
structures. Zhao et al. created structures with 12 base pairs
per turn that only fold correctly in the presence of Dox and
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compared them to structures with a natural twist density of
10.5 base pairs per turn. The increased binding affinity of the
Dox also translates into both a higher loading and a slower
and more sustainable release profile.[28]
Other groups have also reported utilizing DNA origami
structure for Dox intercalation and controlled delivery.
Zhang et al. reported the construction of varying shapes
composed of DNA origami structures and loaded these
structures with Dox to measure the in vitro drug release
and the in vivo antitumor effects (Figure 5B).[62] The shapes
reported were a triangle, a square, and a tube. To test the
accumulation, each structure was loaded with quantum dots
and imaged fluorescently. The triangle shape demonstrated
the highest efficacy of passive tumor targeting out of the
three structures and maintained accumulation levels for 24
hours. Both the square and tube showed higher levels of
off-target accumulation in the liver and kidney. The struc-
tures were stable in serum for 24 hours and demonstrated
slow release properties with 20% released after 8 hours. In
more acidic buffer conditions, which more closely mimic a
tumor microenvironment, the release content was increased.
When tumor-bearing mice were injected with saline, Dox,
Dox/origami structures, and origami structures, the Dox/
origami structures were the most effective at reducing the
tumor size. Earlier experiments reported by this group dem-
onstrated that these nanostructures helped avoid multidrug
resistance that Dox can induce by testing the Dox/origami
structures in MCF 7 Dox resistant cells which were created
by exposing the cells to increasing concentrations of Dox
(Figure 5C).[66]
As mentioned earlier during the course of this review,
effective transfection and uptake of DNA nanostructures can
be difficult and lead to low yield and therapeutic dose due to
their net negative charge. Mikkilä et al. coated DNA origami
structures with cowpea chlorotic mottle virus capsid proteins
to allow for a more effective transfection as the proteins have
a positively charge N-terminus.[48] This helps to combat the
net negative charge of the DNA and allowed the structures
to enter the cell. The DNA origami nanostructures “tem-
plate” the self-assembly of the virus proteins with high yield
through electrostatic binding. The results reported transfec-
tion efficiency higher than Lipofectamine 2000. The proposed
uses for these structures are drug delivery applications.
Another method for utilizing DNA origami for delivery is
covalently linking the small molecule drugs or nanoparticles
to the structure. In 2012, Douglas et al. reported the construc-
tion of logic gated nanorobot with a hexagonal barrel shape
(Figure 5D).[67] This barrel can be loaded with therapeutics
through the use of covalent linkers on the inside. The logic gate,
or “lock”, is a 23 base pair sequence that is antigen activated.
This type of sequence is known as an aptamer and its sensitivity
is directly related to the length of the sequence. In the pres-
ence of the correct antigen, the duplex will bind to the antigen
and allow the barrel to open. The length of the sequence is
tuned such that the risk of spontaneous activation is minimized
(at around 16 bp) whilst the sensitivity is preserved as longer
sequences are much less sensitive. These aptamer-based cues
allow for triggering and targeted delivery based on local envi-
ronments that are specific to certain disease modalities.
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Figure 5. A) Doxorubicin-intercalating nanotubes. Reproduced with permission.[28] Copyright 2012, American Chemical Society. B) Doxorubicin-
intercalating nanostructures. Reproduced with permission.[62] Copyright 2014, American Chemical Society. C) Doxorubicin-intercalating
nanostructures in combating drug resistance. Reproduced with permission.[66] Copyright 2012, American Chemical Society. D) DNA origami
nanorobot. Reproduced with permission.[67] Copyright 2012, AAAS.

3.3. Rolling Circle Approach
Rolling circle amplification (RCA) represents a novel way
to approach precision DNA nanostructure construction.
RCA creates long stranded structures with repeating DNA
sequences through the use of a circular template and DNA
polymerase, which copies the template thousands of times.[68]
This allows for the precise templating of the RCA strand. The
polymerase continually adds complementary nucleotides to the
template until it is denatured or runs out of available nucleo-
tides. This creates long stranded DNA with a known, repeating
sequence that is dependent on the sequence of the circular
template.[49] This long stranded DNA can be manipulated
through staple strands similarly to DNA origami.[43,69] The
staple strands can also be manufactured through RCA and
cleaved through a site-specific cleavage. RCA has been used

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 reviews
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Figure 6. A) DNA origami using rolling circle amplification. Reproduced with permission.[69] Copyright 2013, Wiley-VCH. B) RNAi microsponge.
Reproduced with permission.[73] Copyright 2012, Nature Publishing Group. C) DNA nanobelts loaded with reporter/therapeutic molecules and cell
penetrating peptides. Reproduced with permission.[71] Copyright 2015, Wiley-VCH.

to create nanotubes,[70] DNA nanoballs.[49] nanobelts that can
intercalate dox and quantum dots,[71] and DNA nanoclews.[29]
It has also been incorporated into DNA logic gates.[72]
RCA can also be applied similarly to DNA origami, as
demonstrated by Ouyang et al. who created a long stranded
DNA template out of RCA instead of using a bacteriophage
DNA (Figure 6A).[69] These structures can easily be folded
by fewer staple strands than DNA origami and can be uti-
lized for therapeutic, drug delivery purposes as they are
easily internalized by cells.[43,69]
Yan et al. has demonstrated a method to integrate nano-
particles and DNA strands produced in rolling circle

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amplification (RCA) utilizing a similar method to Ouyang creation of unique structures with repeating units. These
(Figure 6C).[71] DNAs are grown in-situ on gold nanopar- repeating units are critical in allowing the precise control of
ticle surface, and the repetitive long chains are folded into the nanostructure degradation, through the incorporation of
so-called DNA belts while remaining at the particle surface. regions with known responses to cellular environments, and
Drugs and targeting agents can be loaded on to the DNAs therefore, therapeutic delivery.
by either merging or intercalating mechanism for theranostic
purposes. Merging was used to load quantum dots (QDs)
for cellular imaging and intercalation was used to load Dox. 3.4. DNA Nanoparticles
Incorporation of the QDs and cellular penetration peptides
(CPPs) on the nanoparticle (NP)–DNA conjugate gives QDs Another possible modality for utilizing DNA as a precision
the ability to get into U87 MG cells, while maintaining the drug or gene delivery vehicle is to create nanoparticles
spacing so that QDs are not quenched by CPPs. Incorpora- comprised of the DNA itself (Figure 7), or a DNA
tion of Dox into the structure also shows a 2.5 times higher nanoparticle.[45,74–89] DNA nanoparticles feature controllable
cell-killing efficacy in U87 MG cells than pure Dox. sizes from ≈20 nm to ≈200 nm in diameter. The fabrication of
Another proposed use for RCA is the self-degradable the particles also allows a polyethylene glycol shell for longer
nanoclew reported by Sun et al.[29] The nanoclew is woven circulation time.[80–84] It is also proved facile to functionalize
by RCA and includes DNase to degrade and release a the particle with targeting groups like serpin enzyme complex
therapeutic load inside the cells. To enhance the loading of receptor, SEC.[76]
the anticancer therapeutic Dox, multiple G–C sequences In this approach, DNA nanoparticles that were trans-
were incorporated. The nanoclew is a composite structure formed from complexes of DNA to nanoparticles from
including a polymeric capsule which contains the DNase and 18–25 nm through the use of poly-l-lysine covalently
is degradable by acidic pH, allowing for triggered release by coupled to a synthetic peptide.[74] Longer chain length
the cellular environment (i.e., the acidity of the late stage (53.7 kDa poly-K) gives smaller particles and significantly
endosome) and protecting the Dox until it can have the most more and longer duration of gene expression than short
therapeutic effect. The capsule is positively charged to allow chain (9.7 kDa poly-K) complex. Moreover, shorter peptide
for incorporation into the structure. Finally, these structures chains give less protection from the DNase both in vitro and
were incorporated with folic acid to allow for the targeting in vivo.[77]
of the folate receptors which are overexpressed in many can- This complex allows the gene transfer into the cell via cer-
cers. These structures showed both efficient internalization tain cellular receptors. Cells containing the desired receptors
and efficient nucleic accumulation. Other DNA devices have showed the peak activity of the luciferase that was transfected.
taken advantage of this late stage endosomal pH shift.[30] This receptor-targeting gene delivery can be apply on some
Kim et al. created a DNA nanoball composed of an RCA emphysema patients, who has deficiency of alpha1–antitrypsin
template that was complimentary and
hybridized to two types of antisense oligo-
nucleotides.[49] These were then condensed
by cationic Mu peptides and coated with
hyaluronic acid to target the CD44 recep-
tors. The addition of Mu decreased the
size and increased the stability against
nuclease.
In 2012, Lee et al. reported the con-
struction of an RNAi microsponge that
was synthesized by the self-assembly of
siRNA polymers that are cleavable after
cellular uptake, thus allowing for the pro-
tection of the siRNA until it has been
successfully delivered (Figure 6B).[73] The
RNA is stable in the pleated nanosheets
that form the microsponge as they exist
in the hairpin modality. The long stranded
RNA was produced through RCA but uti-
lizing RNA polymerase instead of DNA
polymerase. These microsponges, when
combined with cationic polyethylenimine
(PEI) were successfully transfected into
cells and showed knock-down of luciferase
expression.
Rolling circle amplification is a very Figure 7. Proposed delivery path for DNA nanoparticles. Reproduced with permission.[79]
integrative process that allows for the Copyright 2002, Springer.

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 reviews
(A1AT), a specific kind of antiprotease, particularly in the
lung.[75] In this treatment, alveolar macrophages (PAM)
which reside within alveoli are targeted and mannose-ter-
minal glycoprotein conjugates are used. Although the gene
showed low expression in the cells, the gene transfer tech-
nique still appears promising once they have found proper
receptor for this particular problem.
This system is also used as a gene therapy for cystic
fibrosis.[78,80–83,85] Utilizing the same serpin enzyme receptor
targeting.[74,76] and condensing the plasmid DNA for the
complementary DNA (cDNA) encoding human cystic
fibrosis transmembrane conductance regulator (CFTR)
with poly-l-lysine, these particles can be used to correct
the chloride transfer defect.[78] These particles restored the
expression of CFTR and of nitric oxide synthase-2 (NOS-2)
in the nasal epithelium where the complexes were admin-
istered even with only 0.15 µg of DNA administered. Sim-
ilar particles, with serpin targeting, that transfected the
LacZ reporter gene showed galactosidase expression but
no correction of the NOS-2 deficit. When these particles
are poly(ethylene glycol)-modified (PEGylated), there is
a higher avoidance of aggregation, higher circulation time,
and effective transfection of the desired gene in the airway
epithelium.[80] They also do not require the targeting ligand.
The toxicity of these PEGylated particles is minimal in vivo,
stable in saline, and have a half-life of 2–4 hours in mouse
serum.[81] These complexes also do not induce an antibody
response when lacking PEG,[84] and when tested in human
subjects, there were no adverse effects from the drug except
in possibly one subject, and 8 out of the 12 subjects expe-
rienced at least partial, if not complete, CFTR chloride
channel function.[83] Repeating dosing, at least three doses
have been demonstrated as possible with the targeting,
were also suggested as a possible to extend the lifetime of
the transgene expression, despite the low levels of antibody
formation to the ligand.[84]
The PEGylated DNPs also showed long-term transgene
expression in central nervous system in mice.[87] An intracer-
ebral injection of DNPs can transfect both neuron and glia,
and the transgene expression stayed active in the striatum
for up to 8 weeks, at least 100-fold greater than naked DNA.
Bioluminescent imaging (BLI) was also used 11 weeks after
injection and significant higher transgene activity by DNPs
than naked DNA was detected.
Other than treatment, these particles are also useful
in real-time imaging system: BLI, positron emission
tomography (PET), and magnetic resonance imaging
(MRI).[86] The efficacy of gene delivery using poly(ethylene
glycol) (PEG)-stabilized DNPs was assessed using BLI of
transgene expression in wild type and cystic fibrosis mouse
models[88] and in long-term tracking of transgene activity in
brain in 2011.[89]
Another kind of DNA nanoparticle includes the combi-
nation of interfering RNA with actual metallic nanoparticles
to increase the density and the uptake of the siRNA.[15]
These particles, reported by Jensen et al., showed significant
down-regulation of their target: the oncoprotein, Bcl2Like12
which inhibits apoptosis, thus allowing for higher cytotoxicity
of the cancer cells.
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www.MaterialsViews.com
3.5. Other DNA Nanostructures
It is also worthwhile to review some other unique DNA nano-
structures that have been used for drug delivery that don’t
fall entirely within any particular category of synthesis. These
methods also possess the potential of shape control, ease of
functionalization of targeting agents, and incorporation of
drugs.
DNA blockcopolymer system was introduced by
Alemdaroglu et al. in 2008 (Figure 8A).[90] The hydrophobic
core made of polypropylene oxide (PPO) is designated for
incorporating hydrophobic drugs like doxorubicin (Figure 8A).
The DNA shell makes it easier for functionalization of tar-
geting groups, in this case, folic acid. The diameter of the final
particles is roughly 10 nm. Human colon adenocarcinoma
(Caco-2) cells were used to test this delivery system. Fluores-
cence labeling showed that the particles are readily uptaken
by the cells and loading of Dox showed efficient cytotoxicity.
DNA can also be used to create hydrogels for targeted
delivery that can be environmentally triggerable by including
aptamers and disulfide linkages.[91,92]
As Hong et al. reported, one can utilize siRNA to create
nanogels through polymeric condensation.[91] These siRNA
modalities contained Y-branched antisense siRNA to act as a
cross-linker for the short stranded sense and antisense siRNA
strand. Three modalities were synthesized: M-siRNA, which
mixed the sense and antisense, DY-siRNA, which mixed the
sense and the antisense strand with the Y-shaped linkers, and
YY-siRNA which combined the Y-shaped sense and anti-
sense strand (Figure 8B). The gels were condensed through
the use of cationic linear polyethylenimine (LPEI) and
without the Y-shaped linker (M-siRNA) condensed particles
were not formed. However,unlike other nanogels discussed
in this review, there were no acidic cleavable modalities and
so the nanogels must be cleaved by RNA enzymes inside
the cell, specifically Dicer which can cleave artificial siRNA
within the cell.
Cho et al. reported the construction of DNA structured
nanofilm with controllable release properties.[93] Three types
of film were assembled from hairpin shaped, Y-shaped, and
X-shaped DNA that contained “sticky ends” and each film was
assembled, layer-by-layer, through the use of poly-l-Lysine
(Figure 8C). Each layer of DNA film contained Dox as an
intercalating agent and demonstrated different release pro-
files, in serum, of the drug depended on the shape of the
DNA used. X-DNA films showed a low burst initial release
with a slower and more controlled release than the other two
films, H-DNA had a larger initial burst release and a faster
release overall. Thus, this modality can control the release of
the doxorubicin based on the type of film.
Y shaped aptamers can also be used to for big multilay-
ered DNA dendrimers (Figure 8D).[94] By designing each
layer of aptamers differently, the DNA dendrimers contain
fluorophores, targeting ligands (sgc8) and anticancer drugs
(Dox) for imaging, cell recognition and drug delivery. This
complex also showed potent toxicity towards target cancer
cells (human T cell acute lymphoblastic leukemia cell line)
while having low side effects for the non-target cells (human
Burkitt’s lymphoma cell line).
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Figure 8. A) DNA blockcopolymer system. Reproduced with permission.[90] Copyright 2008, Wiley-VCH. B) DNA nanogel. Reproduced with
permission.[91] Copyright 2011, American Chemical Society. C) DNA-structured nanofilm synthesized by layer by layer deposition. Reproduced with
permission.[93] Copyright 2014, Nature Publishing Group. D) DNA dendrimer layered particles. Reproduced with permission.[94] Copyright 2015,
Nature Publishing Group.

4. Summary and Future Outlook variety of sizes and shapes have been fabricated either
through oligonucleotide, RCA or origami methods, along
DNA nanotechnology has shown great potential in con- with other hybrid nanostructures. Their advantages and dis-
trolling drug/gene delivery. DNA based particles of a vast advantages are summarized in Table 1. They can be readily

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 reviews
www.MaterialsViews.com
Table 1. Comparison of different DNA nanotechnology approaches. a naturally biodegradable and biocompatible
polymer, DNA shows quite promising perfor-
Approach Advantage Disadvantage mance in certain types of cells and in mouse,[52]
ODN Commercially available short strands; Less structurally stable than origami; In these early studies, there was no antibody
less types of strands involved in no available programmable designing response against DNA nanostructures. However,
structure construction. tool. given the complexity of human body, the influ-
DNA origami Programmable tools (e.g., caDNAno); Safety concern about the virus DNA ence of particle physiochemical properties on
More condensed for intercalation of backbone; hundreds of types of staple renal systems, and the unlikely but essentially
drugs. strands. harmful genome recombination, further inves-
RCA Economic and easy large-scale Lack of precise control in length and tigation on DNA nanostructures in different
fabrication. structure. types of organs are needed for their clinic appli-
DNA nanoparticles Integration of different materials Safety issue regarding combinations cation translation. iii) Undetermined large scale
properties (e.g., polymer and DNA). of materials. production. Compared with polymer and lipid
delivery systems, DNA nanostructures have
functionalized to target specific cells, elongate circulation added cost, given the synthesis challenges. A few methods,
time, respond to environmental cues, and incorporate drug, including PCR, RCA and in-cell production,[95] are used to
gene and imaging agents. Therefore, as demonstrated in this amplify the DNA strands using relatively cheap costs. How-
review, DNA nanotechnology has become a robust platform ever, considering the complexity of DNA nanostructures,
to bring controls in a lot of different aspects into drug and there is still a gap that needs to be filled. To enlarge the
gene delivery. DNA can also be easily functionalized, as production scale, besides what discussed in this review, new
discussed earlier in this review, which allows for the combi- approaches to synthesis DNA strands and DNA nanostruc-
nation of DNA nanostructures with other nanostructures. tures are also needed. We believe that with the further devel-
This can lead to creative solutions for therapeutics such as opment in drug delivery and resolution of scalability issues,
the combination DNA/gold nanoparticle therapy of Jensen DNA nanotechnology can bring a new concept into carrier
et al.[15] DNA can also be situated onto structures that are not systems and generate useful clinic results.
metallic, such as polymeric structures allowing for codelivery.
The examples we discussed demonstrate the wide breadth
of how DNA can be used to create versatile, precision nano-
structures for controllable, targeted delivery of molecular
Acknowledgements
therapeutics and gene therapeutics.
While DNA nanotechnology holds high promise for
C.A. and S.X. contributed equally to this work. This work is sup-
precision nanomedicine, it is still in very early stage. So far
ported by University of California-San Diego (faculty start-up fund).
to the authors’ knowledge, there is no significant clinical
trial in progress regarding DNA nanotechnology. Some key
issues need to be considered prior to its clinical translation.
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Received: July 21, 2015
Revised: September 3, 2015
Published online: January 3, 2016

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